Fiber diameters control osteoblastic cell migration and differentiation in electrospun gelatin.

Defined electrospinning conditions were used to create scaffolds with different fiber diameters to investigate their interactions with osteoblastic MG63 cells. Nonwoven gelatin scaffolds were electrospun with varied fiber diameters to investigate the effect of fiber size and resultant porosity on cell proliferation, viability, migration, and differentiation. The low toxicity solvent acetic acid:ethyl acetate:water ratio and gelatin concentrations were optimized to create small and large diameter fibers. The fiber diameters obtained by this procedure were 110 +/- 40 nm for the small and 600 +/- 110 nm for the large fibers. Cell viability assays showed that MG63 cells grew similarly on both fibers at the early time point (day 3) but preferred the scaffold with large diameter fibers by the later time points (day 5 and day 7). Confocal microscopic imaging showed that MG63 cells migrated poorly (maximum depth of 18 microm) into the scaffold of small diameter fibers, but readily penetrated (maximum depth of 50 microm) into the scaffold of large diameter fibers. Alkaline phosphatase (ALP) assays showed that MG63 cells differentiated on scaffolds made from both diameter fibers. In longer term experiments, MG63 cells differentiated to a greater extent on scaffolds made from small diameter fibers compared to large diameter fibers at days 3 and 7, but the ALP levels were the same for both diameter fibers by day 14. These results indicate that cells can perceive differences in the diameter and resultant pore size of electrospun gelatin fibers and that they process this information to alter their behavior.

Evaluation of cross-linking methods for electrospun gelatin on cell growth and viability.

The creation of a tissue engineering scaffold via electrospinning that has minimal toxicity and uses a solvent system composed of solvents with low toxicity and different cross-linking agents was investigated. First, a solvent system of acetic acid/ethyl acetate/water (50:30:20) with gelatin as a solute was evaluated. The optimum system for electrospinning a scaffold with the desired properties resulted from a gelatin concentration of 10 wt %. Several different methods were used to cross-link the electrospun gelatin fibers, including vapor-phase glutaraldehyde, aqueous phase genipin, and glyceraldehyde, as well as reactive oxygen species from a plasma cleaner. Because glutaraldehyde at high concentrations has been shown to be toxic, we explored other cross-linking methods. Using reactive oxygen species from a plasma cleaner is an easy alternative; however, the degradation reaction dominated the cross-linking reaction and the scaffolds degraded after only a few hours in aqueous medium at 37 °C. Glyceraldehyde and genipin were established as good options for cross-linking agents because of the low toxicity of these cross-linkers and the resistance to dissolution of the cross-linked fibers in cell culture medium at 37 °C. MG63 osteoblastic cells were grown on each of the cross-linked scaffolds. A proliferation assay showed that the cells proliferated as well or better on the cross-linked scaffolds than on traditional two-dimensional polystyrene culture plates.